PLASMID 20, 155-157 (1988)

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Physical and Genetic Map of the IncW Plasmid R388

P~LARAVILAAND FERNANDODELA CRUZ Departamento de Biologia Molecular, Vniversidad de Cantabria. C. Herrera Oria s/n. 3901 I Santander, Spain

Received February 3, 1988; revised June 14, 1988

R388 is a conjugative plasmid of the IncW incompatibility group which confers resistance to sulfonamide and trimethoprim. It has been used largely as a recipient plasmid in studies of transposition because of its small size and because it is devoid of transposable elements. Previous maps showed a concentration of re- striction enzyme sites in a short region of the plasmid molecule, but there were about 20 kb of DNA with no restriction enzyme sites known. We have identified 26 new restriction enzyme sites, for the enzymes AvuI, AccI, BstEII, and SphI, conveniently located all around the plasmid molecule.

The plasmids of the IncW group are broad host range and the smallest in size among the conjugative plasmids studied in Enterobacte- riuceae. R388 is one plasmid of this group (Datta and Hedges, 1972). It is 33 kb long and confers resistance to sulfonamide [determined by a drug-resistant dihydropteroate synthase (Swedberg and Skold, 1983)] and to trimeth- oprim [determined by a methotrexate-resis- tant dihydrofolate reductase of known se- quence (Swift et al., 1981)]. The IncW plas- mids are closely related: when R388, pSa, and R7K (the best known members of this group) were compared, they showed a high degree of homology in the regions encoding replication and transfer functions and showed obvious differences in the regions encoding antibiotic resistance (Ward and Grinsted, 1982). Because of homology between pSa and R388 we as- sume that the replication region of R388 lays on the 1.9-kb DNA segment between coor- dinates 4.50 and 6.40 kb (See Fig. 1) according to the studies in pSa (Tait et al., 1982).

The previous map of R388 (Ward and

Grinsted, 1982) has been expanded in this

work by the addition of site locations for en- donucleases AccI, AM, BstEII, and 5’phI (Fig. 1). All the sites of the previously published map have been rechecked, and the significant differences have been corrected. For instance, we detected an extra 0.3-kb BumHI fragment (coordinates 32.90-0.20 kb) and an extra 0.15 kb HindI fragment (coordinates 1.45- 1.60 kb). The sites for BstEII have been taken from Diaz-Aroca et al. (1984) with only minor modifications.

In Fig. 1, coordinate 0.00 kb was arbitrarily selected for the single Sal1 site in R388, and the numeration goes toward the replication region. When R388 was digested with AvaI, 15 fragments were seen after 1% agarose gel electrophoresis, to which we assigned the let- ters “a” to “0”. The sizes of the fragments are indicated in the legend of Fig. 1. Four more fragments could be visualized when electrophoresis was carried out in 6% poly- acrylamide gels (their sizes were 0.19, 0.09, 0.06, and 0.05 kb). Only the location of the 0.19-kb fragment (Ar, in Fig. 1) has been es- tablished (see below).

The addition of the sizes of all AvuI frag- ments gave a molecular length of 33 kb, which is in agreement with the sum of the 25 BglI + PvuII fragments (4.60,3.55,3.40,3.10,2.10, 1.85, 1.80, 1.50, 1.50, 1.37, 1.28, 1.20, 0.95, 0.80, 0.62. 0.60, 0.58, 0.45, 0.41, 0.28, 0.23, 0.22,0.20,0.15, and 0.10 kb). So the real size of R388 must be very close to 33 kb.

To place the AvuI sites we also used map- ping data obtained with pUB5572 [an R388 deletion derivative consisting of the BglII fragment between coordinates 0.65 and 9.35 kb (Ward and Grinsted, 1978)] and with pSU 1027 [one-ended transposition recombi-

155 0147-619X/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All tights of reproduction in any form reserved

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Tra

FIG. 1. Physical and genetic map of R388. [See Avila (1986) for details.] The inner circle shows the proposed coordinate system for the plasmid (in kb) starting at the single WI site. Restriction endonuckase recognition sites: BglII (L), EcoRI (E), HindI (H), PvuII (P), PtiI (T), Sull (S). In the concentric cimles are marked the sites for AvuI (A), BumHI (B), BstEII (C), SphI (D), and Ad (I) with lowercase letters representing the particular fragment, in decreasing order. Designation and size of the Avd fragments: Aa = 6.90, Ab = 6.90, AC = 3.30, Ad = 3.15, Ae = 2.45, A$= 1.55, Ag 1.51, Ah = 1.48, Ai = 1.21, Aj = 0.97, Ak = 0.7 1, AI = 0.68, Am = 0.66, Ah 0.60, Ao = 0.54, and Ap = 0.19 kb. The outer part of the map shows the location of the known phenotype of R388. Tra = conjugal transfer region (P. Avila, M. L. Llosa, and F. de la Cruz, in preparation), Rep = vegetative DNA replication (assigned by homology with pSa), Su = sulfonamide resistance, Tp = trimethoprim resistance.

nant derived from R388 (Motsch et al., 1985)j. To construct the AvaI map of R388, first we mapped the AvaI sites in pUB5572. From them we knew the positions of the 1.5 l-, 0.97-, 0.66-, and 0.60-kb fragments. The 3.15 and 2.45-kb AvuI fragments could be placed by analysis of the digestion products of R388 with AvuI + Bg!II. Similarly, a AvuI + Sal1 diges- tion allowed us to place the 1.2 1-kb AvuI frag- ment. The 0.19-kb AvuI-p fragment was placed between the 2.45- and the 1.21-kb AvaI frag-

merits Ae and Ai after digestion analysis of the 0.95-kb Sal1 + BstEII fragment isolated from a gel. The fragments of 0.71 and 0.68 kb were placed after a lava1 + BstEII digestion. Analysis of the pSU1027 digestion products allowed us to map the 3.3-kb fragment. The remaining AvaI fragments were placed after determining the localization of the AccI and Sphr sites,

R388 produced three fragments a&r digs tion with Accl: 19.65, 10.05, and 3.30 kb. We

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knew the position of one AccI site (which co- incides with the Sal1 site). The other two were identified by AccI + BMII and AvuI + AccI digestion analysis. We simultaneously located the AccI fragments, the 6.9-, 1.55-, and the 1.48-kb AvuI fragments.

When R388 was digested with SphI, five fragments were obtained: 11.60, 9.10, 5.05, 4.80, and 2.45 kb. Two of these sites flank the 2.45-kb fragment contained in pUB5572. The remaining ones were placed by combination of BstEII + SphI, SphI + AccI, and SphI + AvuI digestion analysis. From these results all the SphI sites were placed and we mapped the rest of the AvuI fragments.

Although the smallest AvuI fragments (0.09, 0.06, and 0.05 kb) have not been mapped, it was possible to know their approximate lo- cation. The 0.09-kb fragment is contained within pUB5572 and its most probable posi- tion is besides the 0.66-kb fragment (Fig. 1, fragment Am). The 0.06- and 0.05-kb fmg- ments could be next to the AccI fragment AC (3.3 kb, Fig. 1) because there is a difference of 0.1 kb between the coordinates of AccI frag- ment (as given in the map) and the actual size of the fragment in agarose gels.

This map of R388 will be a useful tool to study the molecular biology of this plasmid as

well as the products obtained in recombination experiments in which R388 is used as a recip- ient plasmid.

ACKNOWLEDGMENTS

This work was supported by grants from the Comisibn Asesora de Investigacibn Cientifica y T6cnica (3047/83) to F.C. P.A. was supported by a research studentship from the MEC.

REFERENCES

AVILA, P. (1986). “TranspoGci6n monoterminal: Carac- terizacibn gen&ica y mecanismo molecular.” Ph.D. thesis, Universidad Complutense, Madrid.

DATTA, N., AND HEDGES, R. W. ( 1972). J. Gen. Microbial. 72,349-3x

DIAZ-AROCA, E., DE LACRUZ, F., ZABALA, J. C., AND OR- TIZ, J. M. (1984). Mol. Gen Genet 193,493-499.

M~TSCH, S., SCHMITT, R., AVILA, P., DE LA CRUZ, F., WARD, E., AND GRINSTED, J. (1985). Nucl. Acids Rex 13,3335-3342.

SWEDBERG, G., AND SK~LD, 0. (1983). J. Bacterial. 153, 1228-1237.

SWIFT, G., MCCARTHY, B. J., AND HEFFRON, F. (1981). Mol. Gen. Genet. l&441-447.

TAIT, R. C., CLOSE, T. J., RODRIGUEZ, R. L., AND KAM), C. I. (1982). Gene 20,39-49.

WARD, J. M., AND GRINSTED, J. (1978). Gene 3, 87-95. WARD, J. M., AND GRINSTED, J. (1982). Plasmid 7,239-

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Communicated by Stuart B. Levy